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C Cheesman

Publications and source records attributed to C Cheesman.

6 recordsLinked to original sources
PubMed2004

The refolding and reassembly of Escherichia coli heat-labile enterotoxin B-subunit: analysis of reassembly-competent and reassembly-incompetent unfolded states.

The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is an exceptionally stable protein maintaining its quaternary structure over the pH value range 2.0-11.0. Up to 80% yields of reassembled pentamer can be obtained in vitro from material disassembled for very short incubation periods in KCl-HCl, pH 1.0. However, when the incubation period in acid is extended, the reassembly yield decreases to no more than 20% (Ruddock et al. (1996) J. Biol. Chem. 271 19118-19123). Here we demonstrate that the ion species present in the disassembly conditions strongly influence the reassembly competence of EtxB showing that 60% reassembly yields can be achieved, even after prolonged incubations, by the use of a phosphate buffer for acid disassembly. Using this system, we have fully characterized the disassembly and reassembly behavior of EtxB by electrophoretic, immunochemical, and spectroscopic techniques and compared it with that previously observed. Depending on the denaturation system used, the acid-denatured monomer is either in a predominantly reassembly-competent state (H(3)PO(4) system) or in a predominantly reassembly-incompetent conformation (KCl-HCl system). Interconversion between these two conformations in the denatured state is possible by the addition of salts to the denatured protein. The results are consistent with the previous hypothesis that the conversion between reassembly-competent and -incompetent states corresponds to a cis/trans isomerization of a peptide bond, presumably that to Pro93.

Bacterial Toxins↗
PubMed2004

The disassembly and reassembly of mutants of Escherichia coli heat-labile enterotoxin: replacement of proline 93 does not abolish the reassembly-competent and reassembly-incompetent states.

The carrier moiety of heat-labile enterotoxin of Escherichia coli (EtxB) is formed by the noncovalent association of identical monomeric subunits, which assemble, in vivo and in vitro, into exceptionally stable pentameric complexes. In vitro, acid disassembly followed by neutralization results in reassembly yields of between 20% and 60% depending on the identity of the salts present during the acid denaturation process. Loss of reassembly competence has been attributed to isomerization of the native cis-proline residue at position 93. To characterize this phenomenon further, two mutants of EtxB at proline 93 (P93G and P93A) were generated and purified. The proline variants reveal only minor differences in their biophysical and biochemical properties relative to wild-type protein, but major changes were observed in the kinetics of pentamer disassembly and reassembly. Additionally, a loss of assembly competence was observed following longer term acid treatment, which was even more marked than that of the wild-type protein. We present evidence that the loss of assembly competence of these mutants is best explained by a cis/trans peptidyl isomerization of the unfolded mutant subunits in acid conditions; this limited reassembly competence and the biophysical properties of the native P93 mutant pentamers imply the retention of the native cis conformation in the nonproline peptide bond between residues 92 and 93 in the mutated proteins.

Alanine↗
PubMed1999

Selective advantages created by codon ambiguity allowed for the evolution of an alternative genetic code in Candida spp.

Several species of the genus Candida decode the standard leucine CUG codon as serine. This and other deviations from the standard genetic code in both nuclear and mitochondrial genomes invalidate the notion that the genetic code is frozen and universal and prompt the questions 'why alternative genetic codes evolved and, more importantly, how can an organism survive a genetic code change?' To address these two questions, we have attempted to reconstruct the early stages of Candida albicans CUG reassignment in the closely related yeast Saccharomyces cerevisiae. These studies suggest that this genetic code change was driven by selection using a molecular mechanism that requires CUG ambiguity. Such codon ambiguity induced a significant decrease in fitness, indicating that CUG reassignment can only be selected if it introduces an evolutionary edge to counteract the negative impact of ambiguity. We have shown that CUG ambiguity induces the expression of a novel set of stress proteins and triggers the general stress response, which, in turn, creates a competitive edge under stress conditions. In addition, CUG ambiguity in S. cerevisiae induces the expression of a number of novel phenotypes that mimic the natural resistance to stress characteristic of C. albicans. The identification of an evolutionary advantage created by CUG ambiguity is the first experimental evidence for a genetic code change driven by selection and suggests a novel role for codon reassignment in the adaptation to new ecological niches.

Adaptation, Biological↗
PubMed1996

A pH-dependent conformational change in the B-subunit pentamer of Escherichia coli heat-labile enterotoxin: structural basis and possible functional role for a conserved feature of the AB5 toxin family.

The non-covalently associated B-subunit moieties of AB5 toxins, such as cholera toxin and related diarrheagenic enterotoxins, exhibit exceptional pH stability and remain pentameric at pH values as low as 2.0. Here, we investigate the structural basis of a pH-dependent conformational change which occurs within the B5 structure of Escherichia coli heat-labile enterotoxin (EtxB) at around pH 5.0. The use of far-UV CD and fluorescence spectroscopy showed that EtxB pentamers undergo a fully reversible pH-dependent conformational change with a pKa of 4.9 +/- 0.1 (R2 = 0.999) or 5.13 +/- 0.01 (R2 = 0.999), respectively. This renders the pentamer susceptible to SDS-mediated disassembly and decreases its thermal stability by 18 degrees C. A comparison of the pH-dependence of the structural change in EtxB5, with that of a mutant containing a Ser substitution at His 57, revealed that the pKa of the conformational change was shifted from ca. 5.1 to 4.4. This finding suggests that protonation of the imidazole side chain of His 57 might facilitate disruption of a spatially adjacent salt bridge, located between Glu 51 and Lys 91 in each B-subunit, thus triggering the conformational change in the pentameric structure. The pH-dependent conformational change was found to be inhibited when B-subunits bound to monosialoganglioside, GMI; and to have no effect on the stability of interaction between A- and B-subunits within the AB5 complex. This suggests that the conformational change is unlikely to have a direct involvement in toxicity. Conservation of the pH-dependent conformational change in the AB5 toxin family, combined with the potential exposure of the hydrophobic core of beta-barrel in the monomeric units, leads to the proposal that the conformational change may be the common feature that ensures the secretion of these proteins from the Vibrionaceae.

Amino Acid Sequence↗
PubMed1996

Assembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Kinetics and molecular basis of rate-limiting steps in vitro.

The B subunits of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) assemble in vivo into exceptionally stable homopentameric complexes, which maintain their quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. Recently, we showed that the simultaneous protonation of two of the COOH-terminal carboxylates in pentameric EtxB was required to cause its disassembly at pH values below 2.0 (Ruddock, L., Ruston, S. P., Kelly, S. M., Price, N. C., Freedman, R. B., and Hirst, T. R.(1995) J. Biol. Chem. 270, 29953-29958). Here, we investigate the influence of environmental parameters on the kinetics of reassembly of acid-generated EtxB monomers in vitro. Such monomers were found to undergo a further acid-mediated conformational change, with an activation energy of 76 +/- 2 J.mol-1.K-1, consistent with isomerization of the cis-proline residue at position 93, and which prevented subsequent EtxB reassembly. By using rapid neutralization of acid-generated monomers, a high proportion of the B-subunits adopted an assembly-competent conformation, which resulted in up to 75% of the protein reassembling into a stable pentameric complex, indistinguishable from native EtxB pentamers. The rate-limiting step in reassembly, over a concentration range of 50-200 microg/ml, was shown to be due to an intramolecular event, which exhibited a pH dependence with a pKa of 7.0. Modification of EtxB with amine-specific probes revealed that the protonation state of the NH2-terminal alanine residue was responsible for the pH dependence of reassembly. The implications of these findings for the biogenesis of Escherichia coli enterotoxin and related enterotoxins in vivo, are considered.

Administration, Oral↗